(7) PROJECT SUMMARY/ABSTRACT Cytochrome P450 enzymes (CYPs) in humans are a family of heme-containing enzymes involved in metabolism of steroids, vitamins, eiconsanoids, and xenobiotics such as drugs, complex plant alkaloids, carcinogens, and other small molecules. The role of CYPs in metabolism of ~75% of all drugs has made this family of enzymes of considerable interest in medicine and for human health. CYP2D6 is one of the significant drug metabolizing enzymes responsible for metabolism of ~20% of commonly prescribed drugs, including drugs with a narrow therapeutic window. Importantly, the enzyme also displays multiple polymorphic forms that contribute to inter-individual difference in responses to drugs metabolized by CYP2D6. While much work has been performed in examining how amino acid changes near the active site affect activity, significant gaps in knowledge remain in understanding how amino acid changes distal to the active site, such as those frequently found in common polymorphic variants, affect the ability of the enzyme to interact with substrates and lead to alterations in drug metabolism. The project proposed here will contribute to understanding the role of amino acid variations both proximal and distal to the active site in determining CYP2D6 activity in regards to changes in enzyme structure/function. We will take three approaches to our studies: Aim 1: To identify CYP2D6 amino acid variants that affect NADPH coupling. NADPH-P450 reductase is an NADPH-binding protein that donates electrons to CYP2D6 for the monoxygenase reaction. NADPH coupling has been associated with the overall efficiency of substrate metabolism and amino acid variation within the enzyme have been shown to result in significant changes in NADPH coupling. CYP2D6 variants that have altered activity for typical substrates will be examined for changes in NADPH coupling rates. Aim 2: Determine the functions of active site ?gating? residues in the reference variant of CYP2D6 (CYP2D6*1). Our preliminary molecular simulation data indicate that Trp-75 functions as a ?swinging-gate? residue to regulate access to the active site. Site directed mutagenesis coupled with further simulation analysis and in vitro experiments will be performed to determine the role of this amino acid on substrate access/egress and overall binding and metabolism of substrates. Aim 3: Determine how changes in amino acids that line access channel regulate substrate access and regioselectivity of product formation in the CYP2D6*53 variant. In our preliminary data, we show that CYP2D6*53 (which has two amino acid changes: Phe120Ile and Ala122Ser) has a 10-fold increase enzyme activity as compared to reference CYP2D6. The role of these amino acids in regulating substrate access will be examined using in-vitro activity assays, molecular dynamics, and crystallographic methods.